Second Edition December

August 2005
2011

EpiTect® MethyLight PCR Handbook

EpiTect MethyLight PCR Kit

EpiTect MethyLight PCR + ROX™ Vial Kit

For quantitative methylation analysis using

sequence-specific probe-based real-time PCR

Sample & Assay Technologies

QIAGEN Sample and Assay Technologies
QIAGEN is the leading provider of innovative sample and assay technologies, enabling
the isolation and detection of contents of any biological sample. Our advanced,
high-quality products and services ensure success from sample to result.
QIAGEN sets standards in:
I Purification of DNA, RNA, and proteins
I Nucleic acid and protein assays
I microRNA research and RNAi
I Automation of sample and assay technologies
Our mission is to enable you to achieve outstanding success and breakthroughs. For
more information, visit www.qiagen.com.
Contents
Kit Contents 4
Shipping and Storage 4
Product Use Limitations 5
Product Warranty and Satisfaction Guarantee 5
Technical Assistance 5
Safety Information 6
Product Specifications 6
EpiTect MethyLight PCR Kit 6
EpiTect MethyLight PCR + ROX Vial Kit 7
Quality Control 7
Introduction 8
2x EpiTect MethyLight Master Mix 9
MethyLight Assays 10
Equipment and Reagents to Be Supplied by User 13
Important Notes 14
Protocols
I Methylation-Specific Real-Time PCR Analysis Using TaqMan Probes
(ABI 7000, 7300, 7700, 7900HT, StepOne, and StepOnePlus) 15
I Methylation-Specific Real-Time PCR Analysis Using TaqMan Probes
(Applied Biosystems 7500 and Other Instruments) 19
Troubleshooting Guide 24
Appendix A: Working with Bisulfite Converted DNA 28
Appendix B: Calculation of the Methylation Rate with EpiTect MethyLight Assays 30
References 30
Ordering Information 31

EpiTect MethyLight PCR Handbook 08/2011 3

Kit Contents
EpiTect MethyLight PCR Kit (200) (1000)
Cat. no. 59436 59438
Number of reactions (50 µl) 200 1000
2x EpiTect MethyLight Master Mix, containing: 3 x 1.7 ml 25 ml
I HotStarTaq® Plus DNA Polymerase
I EpiTect Probe PCR Buffer
I dNTP mix (dATP, dCTP, dGTP, dTTP)
I ROX passive reference dye
RNase-Free Water 2 x 2 ml 20 ml
Handbook 1 1

EpiTect MethyLight PCR + ROX Vial Kit (200) (1000)

Cat. no. 59496 59498
Number of reactions (50 µl) 200 1000
2x EpiTect MethyLight Master Mix (w/o ROX), 3 x 1.7 ml 25 ml
containing:
I HotStarTaq Plus DNA Polymerase
I EpiTect Probe PCR Buffer
I dNTP mix (dATP, dCTP, dGTP, dTTP)
50x ROX Dye Solution 210 µl 1.05 ml
RNase-Free Water 2 x 2 ml 20 ml
Handbook 1 1

Shipping and Storage

EpiTect MethyLight PCR Kits are shipped on dry ice. The kits should be stored
immediately upon receipt at –20°C in a constant-temperature freezer and protected
from light. When stored under these conditions and handled correctly, this product can
be stored at least until the expiration date (see the inside of the kit lid) without showing
any reduction in performance.

4 EpiTect MethyLight PCR Handbook 08/2011

Product Use Limitations
The EpiTect MethyLight PCR Kits are intended for molecular biological applications. These
products are not intended for the diagnosis, prevention, or treatment of a disease.
All due care and attention should be exercised in the handling of the products. We
recommend all users of QIAGEN® products to adhere to the NIH guidelines that have
been developed for recombinant DNA experiments, or to other applicable guidelines.

Product Warranty and Satisfaction Guarantee

QIAGEN guarantees the performance of all products in the manner described in our
product literature. The purchaser must determine the suitability of the product for its
particular use. Should any product fail to perform satisfactorily due to any reason other
than misuse, QIAGEN® will replace it free of charge or refund the purchase price. We
reserve the right to change, alter, or modify any product to enhance its performance
and design. If a QIAGEN product does not meet your expectations, simply call your
local Technical Service Department or distributor. We will credit your account or
exchange the product — as you wish. Separate conditions apply to QIAGEN scientific
instruments, service products, and to products shipped on dry ice. Please inquire for
more information.
A copy of QIAGEN terms and conditions can be obtained on request, and is also
provided on the back of our invoices. If you have questions about product specifications
or performance, please call QIAGEN Technical Services or your local distributor (see
back cover or visit www.qiagen.com).

Technical Assistance
At QIAGEN, we pride ourselves on the quality and availability of our technical support.
Our Technical Service Departments are staffed by experienced scientists with extensive
practical and theoretical expertise in sample and assay technologies and the use of
QIAGEN products. If you have any questions or experience any difficulties regarding
the EpiTect MethyLight PCR Kit or QIAGEN products in general, please do not hesitate
to contact us.
QIAGEN customers are a major source of information regarding advanced or
specialized uses of our products. This information is helpful to other scientists as well
as to the researchers at QIAGEN. We therefore encourage you to contact us if you
have any suggestions about product performance or new applications and techniques.
For technical assistance and more information, please see our Technical Support
Center at www.qiagen.com/Support or call one of the QIAGEN Technical Service
Departments or local distributors (see back cover or visit www.qiagen.com).

EpiTect MethyLight PCR Handbook 08/2011 5

Safety Information
When working with chemicals, always wear a suitable lab coat, disposable gloves,
and protective goggles. For more information, please consult the appropriate material
safety data sheets (MSDSs). These are available online in convenient and compact PDF
format at www.qiagen.com/support/MSDS.aspx where you can find, view, and print
the MSDS for each QIAGEN kit and kit component.

24-hour emergency information

Emergency medical information in English, French, and German can be obtained
24 hours a day from:
Poison Information Center Mainz, Germany
Tel: +49-6131-19240

Product Specifications
EpiTect MethyLight PCR Kit
2x EpiTect MethyLight Master Mix contains:
HotStarTaq Plus HotStarTaq Plus DNA Polymerase is a modified form of a
DNA Polymerase: recombinant 94 kDa DNA polymerase, originally isolated
from Thermus aquaticus, cloned into E. coli. (Deoxynucleoside-
triphosphate: DNA deoxynucleotidyl-transferase, EC 2.7.7.7).
The enzyme is activated by a 5-minute, 95°C incubation step.
EpiTect Probe PCR Novel PCR buffer for highly sensitive methylation quantification
Buffer of bisulfite converted DNA targets; includes multiplex-
PCR–enabling Factor MP.
dNTP mix: Contains dATP, dCTP, dGTP, and dTTP of ultrapure quality.
ROX passive Optimized concentration of fluorescent dye for normalization
reference dye: of fluorescent signals on instruments from Applied Biosystems
(models 7000, 7300, 7700, 7900HT, StepOne™, and
StepOne Plus™, but not Applied Biosystems® 7500 Real-Time
PCR Systems).
RNase-free water: Ultrapure quality, PCR-grade.

6 EpiTect MethyLight PCR Handbook 08/2011

EpiTect MethyLight PCR + ROX Vial Kit
2x EpiTect MethyLight PCR Master Mix (w/o ROX) contains:
HotStarTaq Plus HotStarTaq Plus DNA Polymerase is a modified form of a
DNA Polymerase: recombinant 94 kDa DNA polymerase, originally isolated
from Thermus aquaticus, cloned into E. coli. (Deoxynucleoside-
triphosphate: DNA deoxynucleotidyl-transferase, EC 2.7.7.7).
The enzyme is activated by a 5-minute, 95°C incubation step.
EpiTect Probe PCR Novel PCR buffer for highly sensitive methylation quantification
Buffer: of bisulfite converted DNA targets; includes multiplex-
PCR–enabling Factor MP
dNTP mix: Contains dATP, dCTP, dGTP, and dTTP of ultrapure quality
50x ROX Dye Separate tube of passive reference dye for normalization of
Solution: fluorescent signals on Applied Biosystems 7500 Real-Time PCR
Systems and, optionally, on instruments from Stratagene; not
required for instruments from Bio-Rad/MJ Research, Cepheid,
Corbett, Eppendorf, and Roche
RNase-free water: Ultrapure quality, PCR-grade

Quality Control
2x EpiTect (See quality-control label inside kit lid for lot-specific values)
MethyLight Master Mix
PCR sensitivity and Sensitivity, reproducibility, and specificity in real-time PCR are
reproducibility assay: tested in parallel 20 µl reactions containing 10-fold dilutions
of nucleic acid template.
HotStarTaq Plus (included in the 2x EpiTect MethyLight PCR Master Mix)
DNA Polymerase: Efficiency and reproducibility in PCR are tested. Functional
absence of exonucleases and endonucleases is tested.
Buffers and reagents (included in the 2x EpiTect MethyLight PCR Master Mix).
EpiTect MethyLight Conductivity and pH are tested.
PCR Buffer:
RNase-free water: Conductivity, pH, and RNase activities are tested.

EpiTect MethyLight PCR Handbook 08/2011 7

Introduction
The determination of the relative prevalence of a particular pattern of methylated CpG
dinucleotides in vertebrates is of particular interest in epigenetics research. MethyLight
assays, probe-based real-time PCR for methylation analysis, are often used for sensitive
quantification of the methylation pattern. Depending on the level used for sequence
discrimination, MethyLight assays can be performed in different formats.
Quantitative MethyLight assays comprise PCR primers that do not overlap any CpG
dinucleotides, the potential DNA methylation sites, and TaqMan® probes or other
dual-labeled probes, which are located on a sequence containing the methylation
sites of interest. The sequence discrimination therefore occurs at the level of probe
hybridization — the methylation-specific probe can anneal to the methylated bisulfite
converted DNA sequence whereas the unmethylation specific probe can bind to the
unmethylated bisulfite converted DNA sequence. The primers used are located on
bisulfite converted sequences without CpG sites.
In the case of EpiTect MethyLight Assays, the methylation specific TaqMan probe
contains FAM™ as 5' reporter dye whereas the unmethylation specific TaqMan probe
is linked to VIC®. Measuring the release of FAM and VIC during real-time PCR is then
used to determine the methylation status (Figure 1), whereby the ratio of measured CT
values with both fluorescence dyes allows quantification of the methylation.
Semiquantitative MethyLight assays use methylation-specific primers in conjunction
with a probe located in between. Thus, sequence discrimination occurs at the PCR
amplification level. Two separate assays are required, one to determine the amount
of methylated DNA, the other one to determine the amount of unmethylated DNA
(Figure 2).
A mixture of the formats described is available for MethyLight assays. See reference 1.
EpiTect MethyLight PCR Kits are compatible with all MethyLight assay formats, using
dual-labeled sequence-specific probes, providing flexible, rapid, and sensitive probe-
based real-time PCR quantification of methylation status from CpG sites.

The kits are available in 2 formats:

I EpiTect MethyLight PCR Kit: This kit is supplied with a master mix containing
ROX passive reference dye, and is optimized for use with real-time cyclers that
require a high concentration of ROX dye for fluorescence normalization (e.g.,
instruments from Applied Biosystems, but not Applied Biosystems 7500 Real-Time
PCR Systems).

8 EpiTect MethyLight PCR Handbook 08/2011

I EpiTect MethyLight PCR + ROX Vial Kit: This kit is supplied with a master mix that
is free of ROX dye, and also includes a separate solution of ROX dye which the user
can add to reactions, depending on the real-time cycler used. The kit is intended
for use with cyclers that require a lower concentration of ROX dye for fluorescence
normalization (e.g., Applied Biosystems 7500 Real-Time PCR Systems), for use with
cyclers that allow optional use of ROX dye (e.g., instruments from Stratagene), and
for use with cyclers that do not require ROX dye. Running reactions without ROX
dye allows greater flexibility when choosing reporter dyes for probes.

2x EpiTect MethyLight Master Mix

The components of the 2x EpiTect MethyLight Master Mix include HotStarTaq Plus DNA
Polymerase, EpiTect MethyLight PCR Buffer, and ROX passive reference dye (see
descriptions below). The 2x EpiTect MethyLight Master Mix (w/o ROX) contains
HotStarTaq Plus DNA Polymerase and the EpiTect MethyLight PCR Buffer, but no ROX
passive reference dye. The optimized master mixes ensure that the PCR products in a
reaction with bisulfite converted DNA as starting material are amplified with high
efficiency and sensitivity.

HotStarTaq Plus DNA Polymerase

HotStarTaq Plus DNA Polymerase is a modified form of QIAGEN Taq DNA Polymerase,
and is provided in an inactive state and has no enzymatic activity at ambient
temperature. This prevents the formation of misprimed products and primer–dimers
during reaction setup and the first denaturation step. Competition for reactants by PCR
artifacts is therefore avoided, enabling high PCR specificity and accurate quantification.
The enzyme is activated at the start of a reaction by a 5-minute, 95°C incubation step.
The hot start enables reactions to be set up rapidly and conveniently at room temperature.

2x EpiTect MethyLight Master Mix

The 2x EpiTect MethyLight Master Mix has been specifically developed for highly sen-
sitive detection of methylated and unmethylated DNA using sequence-specific probes.
In addition to various salts and additives, the buffer also contains a specially optimized
combination of KCl and (NH4)2SO4, which promotes a high ratio of specific to
nonspecific primer and probe binding during the annealing step of each PCR cycle,
allowing discriminative hybridization of the probes detecting unmethylated and/or
methylated target sequences. The created stringent primer annealing conditions lead to
increased PCR specificity when amplifying bisulfite converted DNA combined with
reliable detection of the methylation degree. When using this buffer, primer annealing
is only marginally influenced by MgCl2 concentration so optimization by titration of
Mg2+ is usually not required. The buffer also contains the synthetic Factor MP. This
synthetic factor increases the local concentration of primers and probes at the DNA
template and stabilizes specifically bound primers and probes, allowing efficient primer
annealing and extension and supporting discriminative probe hybridization.

EpiTect MethyLight PCR Handbook 08/2011 9

ROX passive reference dye
For certain real-time cyclers, the presence of ROX passive reference dye in real-time PCR
compensates for non-PCR–related variations in fluorescence detection.
The use of ROX dye is necessary for instruments from Applied Biosystems and is
optional for instruments from Stratagene. However, the presence of ROX dye in the
master mix may limit the capabilities for combination of differentially labeled probes on
some instruments. Therefore, we do not recommend using probes that have ROX or
Texas Red® fluorophore as the reporter dye, since their performance in the presence of
ROX passive reference dye is unpredictable. When performing reactions using probes
labeled with Texas Red, ROX, or other equivalent fluorophore, use a real-time cycler
that does not require ROX dye for fluorescence normalization.
The master mix supplied with the EpiTect MethyLight PCR Kit contains ROX dye at a
concentration that is optimal for instruments from Applied Biosystems (models 7000,
7300, 7700, 7900HT, StepOne, and StepOnePlus, but not Applied Biosystems 7500
Real-Time PCR Systems).
For Applied Biosystems 7500 Real-Time PCR Systems and, optionally, instruments from
Stratagene, ROX dye is required at a lower concentration. This is provided by the
EpiTect MethyLight PCR Kit + ROX Vial, which requires the user to add the supplied
ROX dye solution to reactions.
Instruments from all other suppliers, which do not require ROX dye for fluorescence
normalization, should be used with the EpiTect MethyLight PCR Kit + ROX Vial, which
provides master mix that does not contain ROX dye.

MethyLight Assays
EpiTect MethyLight PCR Kits can be used with all TaqMan probes, dual-labeled probes,
and in all MethyLight assay formats.

Methylation-specific TaqMan probes

Methylation-specific TaqMan probes and dual-labeled probes are methylation-specific
oligonucleotides with a fluorophore and a quencher moiety attached (Figure 1). The
fluorophore is at the 5' end of the probe, and the quencher moiety is usually located at
the 3' end or internally. During the extension phase of PCR, the probe is cleaved by the
5'→3' exonuclease activity of Taq DNA polymerase, separating the fluorophore and
the quencher moiety. This results in detectable fluorescence that is proportional to the
amount of accumulated PCR product.

10 EpiTect MethyLight PCR Handbook 08/2011

 FAM
Fluorophore 1 (FAM) VIC
Fluorophore 2 (VIC) Q
Quencher Cleaved nucleotides

Methylated CpG sites Unmethylated CpG sites

excitation excitation
A C
VIC Q FAM Q

excitation excitation

Primer FAM Q Primer VIC Q

3’ 5’ 3’ 5’

Primer Primer

excitation excitation
B D
excitation emission excitation emission FAM Q
VIC Q

FAM VIC

Primer Q
Primer Q

3’ 5’ 3' 5'

Primer Primer

Figure 1. Principle of TaqMan probes used in conjunction with methylation-specific primers in quantitative,
real-time PCR. In quantitative MethyLight Assays, TaqMan probes are located between primers which are
located at methylation sites. In the case of methylated DNA, (A) primers specific to methylated sequences
anneal to the DNA during the PCR annealing step. During the PCR extension step (B) Taq DNA Polymerase
extends the primer. When the enzyme reaches the TaqMan probe annealed to the target sequence, its 5'→3'
exonuclease activity degrades the probe, resulting in physical separation of the fluorophore from the
quencher. In contrast, methylation-specific primers do not anneal to unmethylated, bisulfite converted DNA (B)
and therefore, the TaqMan probe remains undegraded (D) and the proximity of the fluorophore with the
quencher results in efficient quenching of fluorescence from the fluorophore.

TaqMan probes in conjunction with methylation-specific primers

In the second format of MethyLight Assays, TaqMan probes are used in between
methylation-specific primer sites. Bisulfite converted DNA of unmethylated CpG sites
require a different primer sequence to that of methylated CpG sites. Therefore,
2 different primer pairs are used, one specific for methylated and converted DNA, the
other primer pair specific for unmethylated and converted DNA, which is the principle
of MSP (methylation-specific PCR). If the methylation-specific primer binds to the DNA,
it will be elongated during the extension phase and the 5'→3' exonuclease activity
of Taq DNA Polymerase will lead to the degradation of the primer and the release of
the fluorophore. As the fluorophore is now separated from the quencher moiety, a
fluorescence is detectable (Figure 2).

EpiTect MethyLight PCR Handbook 08/2011 11

 FAM
Fluorophore 1 (FAM) Q
Quencher Cleaved nucleotides

Methylated CpG sites Unmethylated CpG sites

A C
excitation excitation

Primer FAM Q FAM Q

3’ 5’ 3’ 5’

Primer

excitation emission
B D
excitation
FAM

Primer Q FAM Q

3’ 5’ 3’ 5’

Primer

Figure 2. Principle of TaqMan probes used in conjunction with methylation-specific primers in

semi-quantitative, real-time PCR. In semiquantitative MethyLight Assays, a TaqMan probe is located between
primers which are located at the methylation sites. Primers specific for methylated sequences only anneal to
methylated DNA (A) and not to unmethylated DNA (C). During the PCR extension step, the fluorophore is
released and the resulting fluorescence is measured (B). In the case of unmethylated DNA, primer annealing
and extension does not occur and fluorescence is not detected (D). Ideally a separate assay is applied, using
primers specific for converted, unmethylated DNA, which determines the amount of unmethylated sequences.
To quantify the methylation degree, a separate assay determining the DNA quantity is required (not shown in
the figure).

For complete quantification, a separate assay is needed using primers specific to

unmethylated and converted DNA, which measures the amount of unmethylated
sequences.

12 EpiTect MethyLight PCR Handbook 08/2011

Equipment and Reagents to Be Supplied by User
When working with chemicals, always wear a suitable lab coat, disposable gloves,
and protective goggles. For more information, consult the appropriate material safety
data sheets (MSDSs), available from the product supplier.
I Primers and probes should be purchased from an established oligonucleotide
manufacturer. Primers should be of standard quality, and probes should be HPLC
purified. Lyophilized primers and probes should be dissolved in TE buffer to
provide a stock solution of 100 µM; concentration should be checked by
spectrophotometry (for details, see, page 29, “Dissolving primer and probes”).
Primer and probe stock solutions should be stored in aliquots at –20°C. Probe stock
solutions should be protected from exposure to light.
I If available, predesigned and validated EpiTect MethyLight Assays can be used.
See our Ordering Information on page 31.
I Nuclease-free (RNase/DNase-free) consumables: Special care should be taken to
avoid nuclease contamination of all reagents and consumables used to set up PCR
for sensitive detection of DNA and cDNA targets.
I PCR tubes or plates (use thin-walled PCR tubes or plates recommended by the
manufacturer of your real-time cycler).

EpiTect MethyLight PCR Handbook 08/2011 13

Important Notes
Selecting kits and protocols
Select the correct EpiTect MethyLight PCR Kit and protocol to use with your real-time
cycler.
See page 15 for recommended cyclers and the protocol for use with the EpiTect
MethyLight PCR Kit and the ABI PRISM® 7000, Applied Biosystems 7300, ABI PRISM
7700, Applied Biosystems 7900 HT, Applied Biosystems StepOne, and Applied
Biosystems StepOnePlus.
Refer to manufacturer’s instructions for fluorescent dye detection capacity.
See page 19 for recommended cyclers and the protocol for use with the EpiTect
MethyLight PCR + ROX Vial Kit and the Applied Biosystems 7500, iCycler iQ®,
Mx3000P®, Mx3005P®, Mx4000®, Rotor-Gene™ 3000, SmartCycler® II, and other
instruments.
Refer to manufacturer’s instructions for fluorescent dye detection capacity.

No template control (NTC)

All methylation quantification experiments should include an NTC, containing all
the components of the reaction except for the template. This enables detection of
contamination.
To control the specificity of the MethyLight Assay to detect bisulfite converted DNA only,
an initial experiment should be conducted using unconverted human genomic DNA,
which should not be amplified. This DNA is part of the EpiTect PCR Control DNA Set
(see Ordering Information on page 31).

Positive control
In some cases it may be necessary to include a positive control, containing a bisulfite
converted DNA of known methylation status. Therefore, use of the EpiTect PCR Control
DNA Set or a single EpiTect control DNA (e.g methylated or unmethylated human
control DNA, bisulfite converted) is recommended. See page 31 for Ordering
Information.

14 EpiTect MethyLight PCR Handbook 08/2011

Protocol: Methylation-Specific Real-Time PCR Analysis
Using TaqMan Probes (ABI 7000, 7300, 7700,
7900HT, StepOne, and StepOnePlus)

Protocol
This protocol is optimized for use of the EpiTect MethyLight PCR Kit with TaqMan probes
and other dual-labeled probes and real-time cyclers from Applied Biosystems except
Applied Biosystems 7500 Real-Time PCR Systems. For further information, see “ROX
passive reference dye”, page 10.

Important points before starting

I Always start with the cycling conditions specified in this protocol.
I Use the primer and probe concentrations specified in this protocol.
I Check whether your real-time cycler is compatible with the chosen combination
of reporter dyes.
I If using an already established MethyLight assay, use the previously established
primer and probe concentrations in combination with the cycling conditions
specified in this protocol.
I Optimal analysis settings are a prerequisite for accurate quantification data. For data
analysis, you should always readjust the analysis settings (i.e., baseline settings and
threshold values) for analysis of every reporter dye channel in every run.

Things to do before starting

I For ease of use, we recommend preparing for each of your targets a 10x
primer–probe mix containing target-specific primers and probe. A 10x
primer–probe mix for MethyLight PCR analysis consists of 4 µM forward primer,
4 µM reverse primer, and 2 µM probe in TE buffer.

Procedure
1. Thaw the 2x EpiTect MethyLight Master Mix, primer and probe solutions,
RNase-free water, and converted DNA. Mix the individual solutions, and place
on ice.
2. Prepare a reaction mix according to Table 1 (page 16) or Table 2 (page 17).
For ease of use, we recommend preparing for each of your targets a 10x
primer–probe mix containing primers and probe(s).
Note: We strongly recommend starting with the optimized Mg2+ concentration
provided in the 2x EpiTect MethyLight Master Mix.
3. Mix the reaction mix thoroughly, and dispense appropriate volumes into PCR tubes
or the wells of a PCR plate.

EpiTect MethyLight PCR Handbook 08/2011 15

 4. Add template DNA (ⱕ100 ng bisulfite converted DNA) to the individual PCR tubes
or wells.
5. Program the real-time cycler according to Table 3 (page 17) or Table 4 (page 18).
Protocol

Note: Check the real-time cycler’s user manual for correct instrument setup (e.g.,
setting up detection of multiple dyes from the same well). Be sure to activate the
detector for both reporter dyes used. Depending on your instrument, it may also
be necessary to perform a calibration procedure for each of the reporter dyes
before they are used for the first time.

Table 1. Preparing reaction mix for methylation PCR analysis using TaqMan probes

Volume per
Component 50 µl reaction* Final concentration
Reaction mix
2x EpiTect MethyLight Master Mix 25 µl 1x
10x primer–probe mix †
5 µl 0.4 µM forward primer‡
0.4 µM reverse primer‡
0.2 µM probe (each,
when using quantitative
MethyLight assays)§
RNase-free water Variable –
Template DNA Variable ⱕ100 ng/reaction
(added at step 4)
Total volume per reaction 50 µl* –

* If your real-time cycler requires a final reaction volume other than 25 µl, adjust the amount of master mix
and all other reaction components accordingly. If using 384-well plates on the Applied Biosystems
7900HT, use a reaction volume of 25 µl.
†
A 10x primer–probe mix consists of 4 µM forward primer, 4 µM reverse primer, and 2 µM probe (each,
when applying quantitative MethyLight assays) in TE buffer.
‡
A final primer concentration of 0.4 µM is optimal. Depending on the synthesis quality and purification
method used, the optimal concentration may be between 0.1 µM and 0.4 µM.
§
A final probe concentration of 0.2 µM gives satisfactory results in most cases. Depending on the synthesis
quality and purification method used, the optimal concentration may be between 0.1 µM and 0.4 µM.

16 EpiTect MethyLight PCR Handbook 08/2011

Table 2. Preparing reaction mix for quantitative methylation PCR analysis using EpiTect
MethyLight Assays

Volume per

Protocol
Component 50 µl reaction* Final concentration
Reaction mix
2x EpiTect MethyLight Master Mix 25 µl 1x
MethyLight Primer Probe Mix, 10x 5 µl 1x
RNase-free water Variable –
Template DNA
(added at step 4) Variable ⱕ100 ng/reaction
Total volume per reaction 50 µl* –

* If your real-time cycler requires a final reaction volume other than 25 µl, adjust the amount of master mix
and all other reaction components accordingly. If using 384-well plates on the Applied Biosystems
7900HT, use a reaction volume of 25 µl.

Table 3. Cycling conditions for methylation PCR analysis using TaqMan probes

Step Time Temperature Additional comments

Initial PCR activation step 5 min 95°C HotStarTaq Plus DNA
Polymerase is activated by
this heating step
2-step cycling: Important: Optimal
performance is only assured
using these cycling conditions
Denaturation 15 s 95°C
Annealing/Extension 60 s 60°C Combined annealing/
extension step with
fluorescence data collection
Number of cycles 40–45 The number of cycles
depends on the amount of
template DNA

EpiTect MethyLight PCR Handbook 08/2011 17

 Table 4. Cycling conditions for quantitative methylation PCR analysis using EpiTect
MethyLight Assays

Step Time Temperature Additional comments

Protocol

Initial PCR activation step 5 min 95°C HotStarTaq Plus DNA

2-step cycling:
Denaturation
Annealing/Extension
Number of cycles
Polymerase is activated by
this heating step
Important: Optimal
performance is only assured
using these cycling conditions
15 s 95°C
60 s 60°C Combined annealing/
extension step with
fluorescence data collection
40–45 The number of cycles
depends on the amount of
template DNA

6. Place the PCR tubes or plate in the real-time cycler, and start the PCR cycling
program.
7. Perform data analysis.
Before performing data analysis, specify the analysis settings. For each probe,
select the analysis settings (i.e., baseline settings and threshold values). Note that
optimal analysis settings are a prerequisite for accurate quantification data.

18 EpiTect MethyLight PCR Handbook 08/2011

Protocol: Methylation-Specific Real-Time PCR Analysis
Using TaqMan Probes (Applied Biosystems 7500 and
Other Instruments)
This protocol is optimized for use of the EpiTect MethyLight PCR Kit + ROX Vial with
TaqMan probes and other dual-labeled probes on Applied Biosystems 7500 Real-Time
PCR Systems and on real-time cyclers which do not require ROX dye for fluorescence
normalization (e.g., cyclers from Bio-Rad/MJ Research, Cepheid, Corbett, and
Stratagene). For further information, see “ROX passive reference dye”, page 10.

Protocol
Important points before starting
I Always start with the cycling conditions specified in this protocol.
I Use the primer and probe concentrations specified in this protocol.
I Check whether your real-time cycler is compatible with the chosen combination of
reporter dyes.
I If using an already established MethyLight assay, use the previously established
primer and probe concentrations in combination with the cycling conditions spec-
ified in this protocol.
I Optimal analysis settings are a prerequisite for accurate quantification data. For
data analysis, you should always readjust the analysis settings (i.e., baseline
settings and threshold values) for analysis of every reporter dye channel in every
run.
I When using the Applied Biosystems 7500 Fast Real-Time PCR System, select the
Run Mode Standard 7500.

Things to do before starting

I For ease of use, we recommend preparing for each of your targets a 10x
primer–probe mix containing target-specific primers and probe. A 10x
primer–probe mix for MethyLight PCR analysis consists of 4 µM forward primer,
4 µM reverse primer, and 2 µM probe in TE buffer.

EpiTect MethyLight PCR Handbook 08/2011 19

 Procedure
1. Thaw the 2x EpiTect MethyLight Master Mix (w/o ROX), 50x ROX Dye Solution,
primer and probe solutions, RNase-free water, and converted DNA. Mix the
individual solutions, and place on ice.
2. Prepare a reaction mix according to Table 5 (page 21) or Table 6 (page 22).
For ease of use, we recommend preparing for each of your targets a 10x
primer–probe mix containing primers and probe(s).
Note: We strongly recommend starting with the optimized Mg2+ concentration
provided by the 2x EpiTect MethyLight Master Mix (w/o ROX). Due to the hot
Protocol

start, it is not necessary to keep samples on ice during reaction setup or while
programming the real-time cycler.
3. Mix the reaction mix thoroughly, and dispense appropriate volumes into PCR tubes
or the wells of a PCR plate.
4. Add template DNA (ⱕ100 ng bisulfite converted DNA) to the individual PCR tubes
or wells.
5. Program the real-time cycler according to Table 7 (page 22) or Table 8 (page 23).
Note: Check the real-time cycler’s user manual for correct instrument setup (e.g.,
setting up detection of multiple dyes from the same well). Be sure to activate the
detector for both reporter dyes used. Depending on your instrument, it may also
be necessary to perform a calibration procedure for each of the reporter dyes
before they are used for the first time.
When using the Applied Biosystems 7500 Fast Real-Time PCR System, select the
Run Mode Standard 7500.

20 EpiTect MethyLight PCR Handbook 08/2011

Table 5. Preparing reaction mix for methylation PCR analysis using TaqMan probes

Volume per reaction

Component 50 µl* 20 µl*† Final concentration
Reaction mix
2x EpiTect MethyLight 25 µl 10 µl 1x
Master Mix (w/o ROX)
50x ROX Dye Solution‡ 1 µl 0.4 µl 1x

Protocol
10x primer–probe mix§ 5 µl 2 µl 0.4 µM forward primer¶
0.4 µM reverse primer¶
0.2 µM probe (each, when
using quantitative
MethyLight assays)**
RNase-free water Variable Variable –
Template DNA Variable Variable ⱕ100 ng/reaction
(added at step 4)
Total volume per reaction 50 µl* 20 µl*† –

* If your real-time cycler requires a final reaction volume other than 50 µl or 20 µl, adjust the amount of
master mix and all other reaction components accordingly.
†
Refers to the Applied Biosystems 7500 Fast Real-Time PCR System.
‡
For real-time cyclers that do not require ROX dye, add RNase-free water instead.
§
A 10x primer–probe mix consists of 4 µM forward primer, 4 µM reverse primer, and 2 µM probe (each,
when applying quantitative MethyLight assays) in TE buffer.
¶
A final primer concentration of 0.4 µM is optimal. Depending on the synthesis quality and purification
method used, the optimal concentration may be between 0.1 µM and 0.4 µM.
**A final probe concentration of 0.2 µM gives satisfactory results in most cases. Depending on the synthesis
quality and purification method used, the optimal concentration may be between 0.1 µM and 0.4 µM.

EpiTect MethyLight PCR Handbook 08/2011 21

 Table 6. Preparing reaction mix for quantitative methylation PCR analysis using EpiTect
MethyLight Assays

Volume per Volume per

50 µl 20 µl
Component reaction* reaction* Final concentration
Reaction mix
2x EpiTect MethyLight 25 µl 10 µl 1x
Master Mix
Protocol

MethyLight Primer 5 µl 2 µl 1x
Probe Mix, 10x
RNase-free water Variable Variable –
Template DNA Variable Variable ⱕ100 ng/reaction
(added at step 4)
Total volume per reaction 50 µl* 20 µl* –

* If your real-time cycler requires a final reaction volume other than 50 µl or 20 µl, adjust the amount of
master mix and all other reaction components accordingly.

Table 7. Cycling conditions for methylation PCR analysis using TaqMan probes

Step Time Temperature Additional comments

Initial PCR activation step 5 min 95°C HotStarTaq Plus DNA
Polymerase is activated by
this heating step
2-step cycling: Important: Optimal
performance is only assured
using these cycling conditions
Denaturation 15 s 95°C
Annealing/Extension 60 s 60°C Combined annealing/
extension step with
fluorescence data collection
Number of cycles 40–45 The number of cycles
depends on the amount of
template DNA

22 EpiTect MethyLight PCR Handbook 08/2011

Table 8. Cycling conditions for quantitative methylation PCR analysis using EpiTect
MethyLight Assays

Step Time Temperature Additional comments

Initial PCR activation step
2-step cycling:
5 min 95°C HotStarTaq Plus DNA
Polymerase is activated by
this heating step
Important: Optimal
performance is only assured

Protocol
using these cycling conditions
Denaturation 15 s 95°C
Annealing/Extension 60 s 60°C Combined annealing/
extension step with
fluorescence data collection
Number of cycles 40–45 The number of cycles
depends on the amount of
template DNA

6. Place the PCR tubes or plate in the real-time cycler, and start the PCR cycling
program.
7. Perform data analysis.
Before performing data analysis, specify the analysis settings. For each probe,
select the analysis settings (i.e., baseline settings and threshold values). Note that
optimal analysis settings are a prerequisite for accurate quantification data.

EpiTect MethyLight PCR Handbook 08/2011 23

Troubleshooting Guide
This troubleshooting guide may be helpful in solving any problems that may arise. For
more information, see also the Frequently Asked Questions page at our Technical
Support Center: www.qiagen.com/FAQ/FAQList.aspx . The scientists in QIAGEN
Technical Services are always happy to answer any questions you may have about
either the information and protocols in this handbook or sample and assay technologies
(for contact information, see back cover or visit www.qiagen.com ).

Comments and suggestions

No signal, or one or more signals detected late in PCR

a) Wrong cycling conditions Always start with the optimized cycling
conditions specified in the protocols. Be sure that
the cycling conditions include the initial step for
activation of HotStarTaq Plus DNA Polymerase
(95°C for 5 min), and the specified times for
denaturation and annealing/extension.
b) HotStarTaq Plus DNA Ensure that the cycling program includes the
Polymerase not activated HotStarTaq Plus DNA Polymerase activation step
(5 min at 95°C) as described in the protocols.
c) Pipetting error or missing Check the concentrations and storage conditions
reagent of the reagents, including primers, probes, and
template nucleic acid. See Appendix A, page 28
for details on evaluating the concentration of
primers and probes. Repeat the assay.
d) Wrong or no detection step Ensure that fluorescence detection takes place
during the during the combined annealing/
extension step when using TaqMan probes.
e) Primer or probe Use optimal primer concentrations. For details, see
concentration not optimal the individual protocols.
In most cases, a probe concentration of 0.2 µM
gives satisfactory results. Depending on the
quality of your probe, results may be improved
by adjusting probe concentration within the
range of 0.1–0.4 µM.
Check the concentrations of primers and probes
by spectrophotometry (see Appendix A, page 28).

24 EpiTect MethyLight PCR Handbook 08/2011

 Comments and suggestions

f) Mg2+ concentration not The Mg2+ concentration in the 2x EpiTect

optimal MethyLight Master Mixes is already optimized.
Increasing the final Mg2+ concentration by
0.5–1 mM may improve results.
g) Problems with starting Ensure that the DNA is fully converted and
template therefore a suited template for methylation
analysis using conversion-specific primers and
probe(s). We recommend the EpiTect Bisulfite Kit.
Use control DNAs as positive controls.
Check the concentration, storage conditions, and
quality of the starting nucleic acids.
If necessary, make new serial dilutions of template
nucleic acid from the stock solutions. Repeat the
assay using the new dilutions.
Ensure that all reagents, buffers, and solutions
used for purification and dilution of template
nucleic acids are free of nucleases.
h) Insufficient amount of Increase the amount of template if possible. Ensure
starting template that sufficient copies of the target nucleic acids
are present in your sample.
i) Insufficient number of cycles Increase the number of cycles.
j) Probe design not optimal If the amplification reaction was successful, there
may be a problem with the probe. Review probe
design.
k) Wrong detection Ensure that the correct detection channel is
channel/filter chosen activated or the correct filter set is chosen for
each reporter dye. Check whether the chosen
combination of reporter dyes is compatible with
the selected detection channels or filter sets.
l) Fluorescence crosstalk Check that the reporter dyes used in your assay
are suitable for double probe analysis on your
instrument. Run appropriate controls to estimate
potential crosstalk effects.

EpiTect MethyLight PCR Handbook 08/2011 25


m) Wrong cycling conditions
n) Analysis settings
(e.g., threshold and baseline
settings) not optimal
o) Use of (bisulfite untreated –
unconverted) genomic DNA
No linearity in ratio of CT value/crossing point to log of the template amount
a) Template amount too high
b) Template amount too low
Increased fluorescence or CT value for “No Template” control
a) Contamination of reagents
b) Minimal probe degradation,
leading to sliding increase
in fluorescence
Varying fluorescence intensity
a) Contamination of
real-time cycler
b) Real-time cycler no longer
calibrated
26
Comments and suggestions
Always start with the optimized cycling
conditions specified in the protocols. Be sure
that the cycling conditions include the initial
step for activation of HotStarTaq Plus DNA
Polymerase (95°C for 5 min), and the specified
times for denaturation and annealing/extension.
Check the analysis settings (threshold and
baseline settings) for each reporter dye. Repeat
analysis using optimal settings for each reporter
dye.
Primers and probes in MethyLight assays are
designed to prime bisulfite converted DNA only.
Therefore, genomic DNA will not be amplified or
detected. Check suitability of primers and probes
with bisulfite converted control DNA.
When signals are coming up at very early CT
values, adjust the analysis settings accordingly.
Increase template amount if possible. Note that
detection of very low starting copy numbers may
not be in the linear range of a standard curve.
Discard all the components of the multiplex assay
(e.g., master mix, primers, and probes). Repeat
the assay using new components.
Check the amplification plots, and adjust the
threshold settings.
Decontaminate the real-time cycler according
to the manufacturer’s instructions.
Recalibrate the real-time cycler according to the
manufacturer’s instructions.
EpiTect MethyLight PCR Handbook 08/2011
 Comments and suggestions

c) Wavy curve at high template In the analysis settings, reduce the number of
cycles used for background calculation (if your
real-time cycler allows you to do so) or reduce the
amount of template.
d) ABI PRISM 7000: Use the recommended reaction volume of 50 µl,
Uneven curves or high and always use optical adhesive covers to seal
standard deviations plates.

EpiTect MethyLight PCR Handbook 08/2011 27

Appendix A: Working with Bisulfite Converted DNA
Methylation occurs on cytosine residues in vertebrates, especially on CpG dinucleotides
enriched in small regions of DNA. Methylation of CpG islands has been shown to be
associated with gene inactivation and plays an important role in the development of
cancer and cell aging.
The methylation status of a DNA can be determined by PCR only when using sodium
bisulfite converted DNA. Incubation of the target DNA with sodium bisulfite results in
conversion of unmethylated cytosine residues into uracil, leaving methylated cytosines
unchanged. For bisulfite conversion, we recommend the EpiTect Bisulfite Kit, which
allows both conversion and cleanup.
Due to bisulfite conversion, the sequence will change, therefore DNA is no longer
complementary and the primer for unmethylated DNA and methylated DNA differs in
sequence. This can be used as a tool for checking methylation status of special CpGs.

Handling EpiTect MethyLight Assays and primers and probes

The EpiTect MethyLight Assays are developed to discriminate against the methylated
and unmethylated allele in parallel, in one sample.
The ten-fold ready-to-use solution of the primer–probe mix in TE buffer can be directly
used with the EpiTect MethyLight PCR Kit.
The EpiTect MethyLight Assays are developed and optimized for use with the EpiTect
MethyLight PCR Kit. They are functionally validated with this kit.

Handling and storing primers and probes

Guidelines for handling and storing primers and probes are given below. For optimal
results, we recommend only combining primers of comparable quality.

Storage buffer
Lyophilized primers and probes should be dissolved in a small volume of low-salt buffer
to give a concentrated stock solution (e.g., 100 µM). We recommend using TE buffer
(10 mM Tris·Cl, 1 mM EDTA, pH 8.0) for standard primers and probes labeled with
most fluorescent dyes.

Storage
Primers should be stored in sterile, nuclease-free TE buffer in small aliquots at –20°C.
Standard primers are stable under these conditions for at least 1 year. Fluorescently
labeled probes are usually stable under these conditions for at least 6–9 months.
Repeated freeze–thaw cycles should be avoided, since they may lead to degradation.
For easy and reproducible handling of primer–probe sets used in multiplex assays, we
recommend preparing 20x primer–probe mixes, each containing the 2 primers and the
probe for a particular target at the suggested concentrations.

28 EpiTect MethyLight PCR Handbook 08/2011

Dissolving primers and probes
Before opening a tube containing lyophilized primer or probe, centrifuge the tube
briefly to collect all material at the bottom of the tube. To dissolve the primer or the
probe, add the required volume of sterile, nuclease-free TE buffer, mix, and leave
for 20 minutes to allow the primer or probe to completely dissolve. Mix again and
determine the concentration by spectrophotometry as described below.
We do not recommend dissolving primers and probes in water. They are less stable in
water than in TE buffer and some may not dissolve easily in water.

Concentration
Spectrophotometric conversion for primers and probes:
1 A260 unit = 20–30 µg/ml
To check primer concentration, the molar extinction coefficient (ε260) can be used:
A260 = ε260 x molar concentration of primer or probe
If the ε260 value is not given on the data sheet supplied with the primers or probes, it
can be calculated from the primer sequence using the following formula:
ε260 = 0.89 x [(A x 15,480) + (C x 7340) + (G x 11,760) + (T x 8850)]
Example
Concentration of diluted primer: 1 µM = 1 x 10–6 M
Primer length: 24 nucleotides with 6 each of A, C, G, and T bases
Calculation of expected A260: 0.89 x [(6 x 15,480) + (6 x 7340) + (6 x 11,760)
+ (6 x 8850)] x (1 x 10–6) = 0.232
The measured A260 should be within ±30% of the theoretical value. If the measured
A260 is very different to the theoretical value, we recommend recalculating the
concentration of the primers or probes, or having the primers or probes resynthesized.
For probes, the fluorescent dye does not significantly affect the A260 value.

Primer and probe quality

The quality of 18–30mers can be checked on a 15% denaturing polyacrylamide gel;*
a single band should be seen. Please contact QIAGEN Technical Services or your local
distributor for a protocol or visit www.qiagen.com .

Probe quality
The quality of the fluorescent label and the purity of TaqMan probes can be determined
by comparing fluorescence before and after DNase digestion. Incubate probes with
or without 5 units DNase at 37°C for 1 hour. A significant difference in fluorescence
following DNase treatment should be detectable.

* When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective
goggles. For more information, please consult the appropriate material safety data sheets (MSDSs)
available from the product supplier.

EpiTect MethyLight PCR Handbook 08/2011 29

Appendix B: Calculation of the Methylation Rate with
EpiTect MethyLight Assays
Quantitative EpiTect MethyLight Assays enable the direct calculation of the methylation
degree in a sample by taking the threshold cycles determined with each of both dyes:
Signal methylated:
CT (CG) (FAM) – represents the threshold cycle of the CG reporter (FAM channel)
Signal unmethylated:
CT (TG) (VIC) – represents the threshold cycle of the TG reporter (VIC channel)

Percentage of methylation: Cmeth = 100/[1+2(CtCG-CTTG)]%* (see reference 1)

Using the single probe-based MethyLight assay format, another calculation has to be
performed. A commonly used method here is the determination of the PMR (percentage
of fully methylated reference) (2).

References
QIAGEN maintains a large, up-to-date online database of scientific publications utiliz-
ing QIAGEN products. Comprehensive search options allow you to find the articles you
need, either by a simple keyword search or by specifying the application, research
area, title, etc.
For a complete list of references, visit the QIAGEN Reference Database online at
www.qiagen.com/RefDB/search.asp or contact QIAGEN Technical Services or your
local distributor.

Cited references
1. Eads, C.A. et al. (2000). MethyLight: A high-throughout assay to measure DNA Methylation. Nucleic Acid
Res. 28, e 32.
2. Cottrell et al. (2007). Discovery and Validation of 3 novel DNA methylation markers of prostate cancer
prognosis. The Journal of Urology 177, 1753.

30 EpiTect MethyLight PCR Handbook 08/2011

Ordering Information
Product
Related products
EpiTect MethyLight Assays
Hs_PITX2
Hs_PLAU
Hs_ONECUT2
Hs_ ERBB2
Hs_CDKN2A
Hs_TMEFF2
EpiTect Bisulfite Kits — for complete bisulfite conversion and cleanup
of DNA for methylation analysis
EpiTect Bisulfite Kit (48)
EpiTect 96 Bisulfite Kit (2)
EpiTect Control DNA — for evaluation of PCR primers used
for methylation analysis
EpiTect Control DNA,
methylated (100)
EpiTect Control DNA,
unmethylated (100)
EpiTect Control DNA (1000)
EpiTect MethyLight PCR Handbook 08/2011
Contents Cat. no.
10-fold primer TaqMan probe mix, 59930
for real-time MethyLight PCR
10-fold primer TaqMan probe mix, 59931
for real-time MethyLight PCR
10-fold primer TaqMan probe mix, 59932
for real-time MethyLight PCR
10-fold primer TaqMan probe mix, 59934
for real-time MethyLight PCR
10-fold primer TaqMan probe mix, 59933
for real-time MethyLight PCR
10-fold primer TaqMan probe mix, 59935
for real-time MethyLight PCR
48 EpiTect Bisulfite Spin Columns, 59104
Reaction Mix, DNA Protect Buffer,
Carrier RNA, Buffers
2 x EpiTect Bisulfite 96-well Plates, 59110
Reaction Mix, DNA Protect Buffer,
Carrier RNA, Buffers
Methylated and bisulfite converted 59655
human control DNA for
100 control PCRs
Unmethylated and bisulfite converted 59665
human control DNA for
100 control PCRs
Unmethylated human control DNA 59568
for 1000 control PCRs
31
Ordering Information
Product Contents Cat. no.

EpiTect PCR Control Human control DNA set (containing 59695

DNA Set (100)
EpiTect Whole Bisulfitome Kit — for amplification of bisulfite converted
DNA
EpiTect Whole Bisulfitome
Kit (25)
EpiTect Whole Bisulfitome
(100)
EpiTect MethyLight PCR Kit — for real-time quantification of methylation
status
EpiTect MethyLight PCR
Kit (200)
EpiTect MethyLight PCR
Kit (1000)
EpiTect MethyLight PCR
+ ROX Vial Kit (200)
EpiTect MethyLight PCR
+ ROX Vial Kit (1000)
both bisulfite converted methylated
and unmethylated DNA and
unconverted unmethylated DNA)
for 100 control PCRs
REPLI-g® Midi DNA Polymerase, 59203
EpiTect WBA Reaction Buffer,
Nuclease-Free Water for 25 whole
bisulfitome amplification reactions
REPLI-g Midi DNA Polymerase, EpiTect 59205
WBA Reaction Buffer, Nuclease-Free
Water for 100 whole bisulfitome
amplification reactions
Master Mix for methylation-specific 59436
real-time PCR analysis, 200 x 50 µl
reactions
Master Mix for methylation-specific 59438
real-time PCR analysis, 1000 x 50 µl
reactions
Master Mix without ROX for 59496
methylation-specific real-time PCR
analysis, 200 x 50 µl reactions
Master Mix without ROX for 59498
methylation-specific real-time PCR
analysis, 1000 x 50 µl reactions

For up-to-date licensing information and product-specific disclaimers, see the respective
QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are
available at www.qiagen.com or can be requested from QIAGEN Technical Services
or your local distributor.

32 EpiTect MethyLight PCR Handbook 08/2011

Notes

EpiTect MethyLight PCR Handbook 08/2011 33

Notes

34 EpiTect MethyLight PCR Handbook 08/2011

Trademarks: QIAGEN®, EpiTect®, HotStarTaq®, Rotor-Gene®, REPLI-g® (QIAGEN Group); ABI PRISM®, Applied Biosystems®,
StepOne™, StepOnePlus™, VIC® (Applera Corporation or its subsidiaries); iCycler iQ® (Bio-Rad Laboratories, Inc.); FAM™, ROX™
(Life Technologies Corporation); TaqMan® (Roche Group); Mx3000P®, Mx3005P®, Mx4000® (Stratagene); SmartCycler®
(Cepheid), Texas Red® (Molecular Probes, Inc.).
Registered names, trademarks etc. used in this document, even when not specifically marked as such, are not to be considered
unprotected by law.
NOTICE TO PURCHASER - LIMITED LICENSE
A license under U.S. Patents 5,786,146, 6,017,704, 6,265,171 and 6,200,756 or their foreign counterparts, owned by The
Johns Hopkins University, is required to practice methylation-specific polymerase chain reaction and related processes (“MSP”)
described in said patents. The purchase price of this product (EpiTect MethyLight PCR Kit) includes limited, nontransferable rights
to use only this amount of the product to practice MSP and related processes described in said patents solely for the internal research
activities of the purchaser. No right to any other application, including any in vitro diagnostic application, or to perform or offer
commercial services of any kind using MSP, including without limitation reporting the results of purchaser's activities for a fee or
other commercial consideration, is hereby conveyed or granted by implication or estoppel. Further information on obtaining
licenses to practice MSP may be directed to the Licensing Department, OncoMethylome Sciences, 2505 Meridian Parkway, Suite
310, Durham, NC 27713.
NOTICE TO PURCHASER: LIMITED LICENSE
For Applicable Countries
Use of this product (EpiTect MethyLight PCR Kit) is covered by one or more of the following US patents and corresponding patent
claims outside the US: 6,127,155, 5,677,152 (claims 1 to 23 only), 5,773,258 (claims 1 and 6 only), and claims outside the US
corresponding to expired US Patent No. 5,079,352. The purchase of this product includes a limited, non-transferable immunity
from suit under the foregoing patent claims for using only this amount of product for the purchaser's own internal research. No
right under any other patent claim and no right to perform commercial services of any kind, including without limitation reporting
the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by
estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further
information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln
Centre Drive, Foster City, California 94404, USA.
The purchase of this product (EpiTect MethyLight PCR Kit) includes a limited, non-transferable right to use the purchased amount of
the product to perform Applied Biosystems’ patented Passive Reference Method for the purchaser’s own internal research. No right
under any other patent claim and no right to perform commercial services of any kind, including without limitation reporting the
results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel.
This product is for research use only. For information on obtaining additional rights, please contact outlicensing@lifetech.com or
Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.
Limited License Agreement
Use of this product signifies the agreement of any purchaser or user of the EpiTect MethyLight PCR Kits to the following terms:
1. The EpiTect MethyLight PCR Kits may be used solely in accordance with the EpiTect MethyLight PCR Handbook and for
use with components contained in the Kit only. QIAGEN grants no license under any of its intellectual property to use or
incorporate the enclosed components of this Kit with any components not included within this Kit except as described in the
EpiTect MethyLight PCR Handbook and additional protocols available at www.qiagen.com.
2. Other than expressly stated licenses, QIAGEN makes no warranty that this Kit and/or its use(s) do not infringe the rights of
third-parties.
3. This Kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.
4. QIAGEN specifically disclaims any other licenses, expressed or implied other than those expressly stated.
5. The purchaser and user of the Kit agree not to take or permit anyone else to take any steps that could lead to or facilitate
any acts prohibited above. QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court, and shall
recover all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement or
any of its intellectual property rights relating to the Kit and/or its components.
For updated license terms, see www.qiagen.com.
© 2008–2011 QIAGEN, all rights reserved.
www.qiagen.com

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